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Articleshttps://doi.org/10.1038/s41556-018-0089-0
1Department of Molecular, Cell and Developmental Biology,
University of California Los Angeles, Los Angeles, CA, USA.
2Molecular Biology Institute, University of California, Los
Angeles, CA, USA. 3Department of Anatomy and Developmental Biology,
Monash University, Clayton, Victoria, Australia. 4Development and
Stem Cells Program, Monash Biomedicine Discovery Institute,
Clayton, Victoria, Australia. 5Australian Regenerative Medicine
Institute, Monash University, Clayton, Victoria, Australia. 6Eli
and Edythe Broad Center of Regenerative Medicine and Stem Cell
Research, University of California Los Angeles, Los Angeles, CA,
USA. 7Howard Hughes Medical Institute, University of California Los
Angeles, Los Angeles, CA, USA. Present address: 8Department of
Biochemistry, McGill University, Montreal, Quebec, Canada. *e-mail:
[email protected]; [email protected]
The broad contours of pre-implantation development are
con-served between mice and humans1. After fertilization to create
the zygote, the embryo undergoes cell divisions, compacts to form
the morula, then undergoes further cell division and cavita-tion to
form the fluid-filled blastocyst. At this point, the first three
cell types—trophoblast, primitive endoderm and epiblast—are
specified, with the epiblast destined to give rise to all embryonic
tis-sues. Upon implantation, the epiblast undergoes dramatic
changes in gene expression and epigenetic state, priming it to
differentiate rapidly in response to external cues. As such, the
epiblast transi-tions from the naive pluripotent state to the
primed pluripotent state. Gastrulation then occurs and pluripotency
is lost altogether.
Despite this similar overall program, it has become clear that
there are dramatic molecular differences between mouse and human
embryo development2–8. However, given the significant lim-itations
in research using human embryos, it has not been possible to
rigorously compare the murine and human naive epiblast.
The traditional approach for deriving and culturing human
embryonic stem cells (hESCs) from pre-implantation embryos results
in cells with primed pluripotency similar to murine
post-implantation epiblast stem cells (EpiSCs). However, new medium
formulations for transitioning or deriving hESCs in the naive state
have now been developed9,10. Critically, naive hESCs largely
recapitulate the transcriptional and epigenetic program of human
pre-implantation epiblast cells6,11,12. At present, naive and
primed hESCs are the only human cell-based models for understanding
the critical fate transition between naive and primed pluripo-tency
in the human embryo and the contrast between murine and human
epiblast.
ResultsActivator protein-2 motifs are strongly enriched in
naive-specific regulatory elements. To identify transcription
factors critical for naive human pluripotency, we mapped open
chromatin using the assay for transposase-accessible chromatin
(ATAC-seq13) in naive and primed hESCs (Supplementary Fig. 1a and
Supplementary Table 1). Cells were cultured in five inhibitors plus
LIF, Activin A and FGF2 (5iLAF) to recapitulate the naive state and
with FGF2 and knockout serum replacement media (KSR) to
recapitulate the primed state9,12. As expected, we observed strong
enrichment of open chromatin at gene promoters (Supplementary Fig.
1b), with enrichment associating with gene expression. We defined
sets of ATAC-seq peaks in naive and primed hESCs, as well as peaks
spe-cific to either the naive or primed states (Supplementary Fig.
1c, Supplementary Table 2 and Methods). Although all sets showed
enrichment of the promoter sequence, this enrichment was much
weaker for naive and primed-specific open sites (Supplementary Fig.
1c), consistent with the general trend that enhancer utilization
rather than promoter openness is more variable between different
cell types14,15.
Broadly, we observed a strong correlation between the
appear-ance of naive-specific ATAC-seq peaks near a gene
transcription start site (TSS), and upregulation of that gene in
the naive state, and between the appearance of a primed-specific
ATAC peak near a gene TSS and downregulation in the naive state
(Fig. 1a,b and Supplementary Fig. 1d,e,f). This was true whether
the ATAC peak was upstream or downstream of the gene transcription
start site (Supplementary Fig. 1e,f). For example, naive-specific
ATAC peaks are observed in the vicinity of the naive-specific
Kruppel-like
TFAP2C regulates transcription in human naive pluripotency by
opening enhancersWilliam A. Pastor1,8, Wanlu Liu1,2, Di Chen1,
Jamie Ho1, Rachel Kim1, Timothy J. Hunt1, Anastasia Lukianchikov1,
Xiaodong Liu3,4,5, Jose M. Polo 3,4,5, Steven E. Jacobsen
1,6,7* and Amander T. Clark 1,6*
Naive and primed pluripotent human embryonic stem cells bear
transcriptional similarity to pre- and post-implantation epiblast
and thus constitute a developmental model for understanding the
pluripotent stages in human embryo development. To identify new
transcription factors that differentially regulate the unique
pluripotent stages, we mapped open chromatin using ATAC-seq and
found enrichment of the activator protein-2 (AP2) transcription
factor binding motif at naive-specific open chromatin. We
determined that the AP2 family member TFAP2C is upregulated during
primed to naive reversion and becomes widespread at naive-specific
enhancers. TFAP2C functions to maintain pluripotency and repress
neuroectodermal differentiation during the transition from primed
to naive by facilitating the opening of enhancers proximal to
pluripotency factors. Additionally, we identify a previously
undiscovered naive-specific POU5F1 (OCT4) enhancer enriched for
TFAP2C binding. Taken together, TFAP2C establishes and maintains
naive human pluripotency and regulates OCT4 expression by
mechanisms that are distinct from mouse.
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All rights reserved.
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Articles NATure Cell BiOlOgy
factor 5 (KLF5), and primed-specific ATAC-seq peaks are observed
in the vicinity of the primed-specific genes ZIC2 and ZIC5 (Fig.
1c,d). These observations are consistent with a high propor-tion of
ATAC-seq peaks corresponding to enhancers that regulate nearby
genes. A comparison with data from a published chromatin
immunoprecipitation assay with sequencing (ChIP-seq) in naive and
primed hESCs16 revealed enrichment of Mediator over naive and
primed-specific ATAC-seq peaks in the corresponding cell type, and
we observed strong enrichment of H3K27Ac at the boundaries
of these peaks, with a dip in the middle probably explained by
nucleosome depletion (Fig. 1e). Mediator and H3K27Ac enrich-ment
are predictive features of active enhancers17,18, further
validat-ing the ATAC-seq peaks as regulatory elements.
To identify transcription factors critical for the activity of
enhancers in the naive and primed states, we determined enrich-ment
of known transcription factor binding motifs in the naive and
primed-specific ATAC peaks (Fig. 1f,g)19. The strongest
sta-tistical enrichment in the naive state corresponded to the
KLF
–1,000 –500 500 1,000
Rel
ativ
e A
TA
C e
nric
hmen
t
–1,000 –500 500 1,000
e
a b
Upregulatedin naive
Downregulatedin naive
c d
Naive(ATAC)
Primed(ATAC)
Chr13:73,600 kb 73,700 kb73,650 kb
KLF5
Naive(ATAC)
Primed(ATAC)
Chr13:100,600 kb 100,660 kb100,630 kb
ZIC5 ZIC2
Motif
AP2
KLF
OCT4
Motif%
Targets%
Background
ZIC
36.49 14.02 P < 10–176SOX
OCT4
22.48 8.99 P < 10–92
14.05 2.75 P < 10–138
Fold enriched
2.60
2.50
5.11
SOX
%Targets
51.49
42.03
11.96
27.33
%Background
16.47
12.88
3.11
14.23
P value
P < 10–708
P < 10–572
P < 10–169
P < 10–128
Foldenriched
3.12
3.26
3.85
1.92
P value
f g
1
2
3
4
5
60
Differential expression of genes binned by distancefrom
naive-specific ATAC peaks (5,032 peaks)
Differential expression of genes binned by distancefrom
primed-specific ATAC peaks (2,562 peaks)
40
20
0
200–
500k
100–
200k
50–1
00k
25–5
0k
10–2
5k
5–10
k2–
5k1–
2k
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ArticlesNATure Cell BiOlOgya
d e
hgf
i
−5,000 bp ATAC peaksummit
Blastocyst overtrophoblast specific genes
Naive hESC overtrophoblast-specific genes
Indi
vidu
alna
ive-
spec
ific
AT
AC
pea
ks
5,000 bp
Rel
ativ
e A
TA
C e
nric
hmen
t
1
2
3
4
Blastocyst ATAC overnaive-specific ATAC peaks
Blastocyst ATAC overprimed-specific ATAC peaks
−5,000 bp ATAC peaksummit
5,000 bp
b c
Blastocyst
Naive hESC
Primed hESC
58,394
19,096
26,712
17,728
5,632
13,779
35,456
GATA3
AP2
KLF
OCT-SOX
2.64
2.35
1.65
1.24
1.12
1.99
2.47
7.80
No enrich
No enrich
1.07
8.47
No enrich
No enrich
1.23
8.03
1.96
2.50
2.32
4.27
1.42
1.11
1.14
3.52
1.09
1.08
1.44
4.43
Gene body
−1,000 TSS 33%
Blas
tocy
st
Naive
hES
C
Prim
ed h
ESC
Naive
prim
ed
inter
sect
(hES
C)
Tripl
e int
erse
ct
Naive
+blas
t inte
rsec
t
(pre
-impla
nt)
Prim
ed b
last
inter
sect
66% TES 1,000
Gene body
−1,000 TSS 33% 66% TES 1,000
Blastocyst over all genes
Blastocyst overepiblast specific genes
Naive hESC overepiblast-specific genes
1
2
3
4
1
2
3
4
5
6Naive hESC over all genes
Rel
ativ
e A
TA
C e
nric
hmen
t
Rel
ativ
e A
TA
C e
nric
hmen
t
Naive(ATAC)
Primed(ATAC)
Blastocyst(ATAC)
Chr12:7,935 kb 7,940 kb 7,945 kb
NANOG
Naive(ATAC)
Primed(ATAC)
Blastocyst(ATAC)
Chr10:8,080 kb 8,120 kb 8,160 kb
GATA3
Blastocyst
3,518
192
1,514
109,668
2,370
0 0.25
Blastocyst ATAC overnaive-specific ATAC peaks
0.5
Naive specificpeaks
Primed specificpeaks
0.75 1.0
Relative enrichment
Fig. 2 | Most naive-specific ATAC peaks are present in other
naive human cells and the human embryo. a, Normalized ATAC-seq
reads from the human blastocyst plotted relative to naive-specific
and primed-specific peaks. Note the far greater enrichment over
naive-specific peaks. b, Blastocyst ATAC-seq plotted relative to
all naive-specific ATAC peaks. Note the enrichment over almost all
naive-specific peaks, indicating that they are open in the
blastocyst. c, Most naive-specific ATAC-seq peaks overlap with a
blastocyst-ATAC peak, but most primed-specific peaks do not. d,e,
ATAC-seq signal for primed hESCs, naive hESCs and blastocyst in the
viscinity of NANOG (d) and GATA3 (e). Peak height is normalized to
the total number of reads in each sample. f,g, Metaplot of ATAC-seq
read density over the gene bodies of 100 genes most highly specific
to trophoblast or epiblast, as defined from single-cell RNA-seq
data in human3, as well as all genes. TES, transcription end site.
h, Venn diagram showing overlap of all ATAC-seq peaks in
blastocyst, naive hESCs and primed hESCs. i, Enrichment of GATA,
AP2, KLF and OCT-SOX motifs in each set identified in h. Note the
enrichment of AP2 and KLF motifs in both blastocyst and naive
hESCs, stronger enrichment of GATA in blastocyst, and stronger
enrichment of OCT-SOX in ESCs.
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NATuRE CELL BioLogy | VOL 20 | MAY 2018 | 553–564 |
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Articles NATure Cell BiOlOgy
motif, consistent with the strong upregulation of KLF family
fac-tors in naive hESCs and the known role for KLF in naive-state
pluripotency in mouse and human9,10,20. Similarly, the motif of the
primed-specific21 ZIC factors was enriched in the primed peaks.
Unexpectedly, very strong enrichment for the activator protein-2
(AP2) transcription factor motif was observed for naive-specific
open chromatin. AP2 transcription factors have been implicated in a
number of developmental processes in mice, including pla-cental
development22–24, neural crest development25 and ectoder-mal
patterning25–27, but are completely dispensable for murine epiblast
formation and mouse pluripotent cell survival22–24,28. Hence, there
may be a human-specific role for an AP2 factor in the naive
state.
Naive-specific regulatory elements are present in vivo. To
deter-mine the in vivo relevance of our set of naive-specific ATAC
peaks,
we performed ATAC-seq on eight pooled pre-implantation human
blastocysts (Supplementary Table 3). We found dramatically
increased openness in the human blastocyst over naive-specific
peaks, both relative to the surrounding sequence and relative to
primed peaks (Fig. 2a-c and Supplementary Fig. 2a), validating the
biological relevance of these peaks. Nonetheless, there were marked
differences between the open chromatin patterns in whole
blasto-cyst and naive hESCs. We reasoned that this was because the
day 6 human blastocyst consists primarily of trophoblast, with a
much smaller fraction of epiblast and hypoblast29. For example, we
found that blastocyst showed lower ATAC-seq enrichment in the
vicinity of the epiblast-specific gene NANOG but higher enrichment
in the vicinity of the trophoblast-specific GATA3 (Fig. 2d,e). This
trend was apparent when we plotted ATAC enrichment over epiblast
and trophoblast-specific gene bodies as defined from published
RNA-seq data (Fig. 2f,g).
10
20
30
40 NaivePrimed
RP
KM
a
TFAP2C
H3
Naive Primed30k 15k 30k 15kCells:
b Chr20:32,040 kb 32,100 kb32,070 kb
CBFA2T2
NaiveATAC
PrimedATAC
PrimedTFAP2C
c
BlastocystATAC
−2,000 −1,000 ATAC peaksummit
1,000 2,000
1
2
3
4
Rel
ativ
e C
hIP
enr
ichm
ent
d TFAP2C ChIP over allnaive-specific ATAC peaks(5,032 peaks)
TFAP2C ChIP overnaive-specific ATAC peakswith AP2 sites)(2,296
peaks)
TFAP2C ChIP overnaive-specific ATAC peakswithout AP2
sites)(2,736 peaks)
(ChIP input dashed lines)
e
−2,000 −1,000 ATAC peaksummit
1,000 2,000
Rel
ativ
e A
TA
C e
nric
hmen
t
Naive ATAC overnaive-specific ATAC peaks
Naive ATAC overnaive-primed intersect
−2,000 −1,000 ATAC peaksummit
1,000 2,000
Rel
ativ
e C
hIP
-seq
enr
ichm
ent
TFAP2C ChIP overnaive-specific ATAC peaks
TFAP2C ChIP overnaive-primed intersect
f
1
2
3
4
5
6
1
2
3
4
(Dashed lines indicate primed ATAC over same peak set)(Dashed
lines indicate ChIP input over same ATAC peak)5 7
PrimedinputNaiveTFAP2CNaiveinputPrimedH3K4me3NaiveH3K4me3PrimedH3K27AcNaiveH3K27Ac
50 kDa
TFAP
2E
TFAP
2D
TFAP
2C
TFAP
2B
TFAP
2A
TFAP
2E
TFAP
2D
TFAP
2C
TFAP
2B
TFAP
2A
Fig. 3 | TFAP2C is highly enriched over naive-specific open
chromatin in humans. a, TFAP2C is highly expressed in naive cells,
both relative to other AP2 transcription factors and relative to
primed cells. Mean and standard deviation are shown, with dots
representing each replicate (n = 4 independent experiments). b,
TFAP2C protein is highly upregulated in the naive pluripotent
state. Data represent one out of five independent experiments with
similar results. H3, histone 3. c, Strong co-enrichment of TFAP2C
with naive-specific ATAC peaks at the CBFA2T2 locus. d, Global
enrichment of TFAP2C relative to the summits of different
categories of naive-specific ATAC peaks. TFAP2C is enriched over
naive-specific ATAC peaks, especially those with AP2 motifs. e,f,
TFAP2C is strongly enriched over naive-specific ATAC peak summits
compared with enrichment over regions that show ATAC enrichment in
both naive and primed cells (naive-primed intersect) (e), even
though both peak sets show similar ATAC enrichment (f). Uncropped
western blot images are provided in Supplementary Fig. 9. Source
data for a are provided in Supplementary Table 8.
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ArticlesNATure Cell BiOlOgya
Control TFAP2C –/– line 1
f
TFAP2C
H3
b
Prim
ed
Naive
stea
dy st
ate
d1 5
iLAF
Prim
ed
d3 5
iLAF
Prim
ed
d5 5
iLAF
Prim
ed
g h
TFAP
2COC
T4
NANO
GSO
X2KL
F4KL
F5
TFCP
2L1
DNM
T3L
SOX1PA
X6ZI
C10.01
0.1
1
10
100
Exp
ress
ion
TF
AP
2C–/
– / W
T
Corepluripotency
Naivepluripotency
Neural
Prim
ed
d5
5iLAF 5iLAF
Passage 1 (d9) Passage 1 (d9)
Passage 2 (d14) Passage 2 (d14)
d14
d19 SOX1
PAX6
H3
OCT4
NANOG
H3
Cont
rol
TFAP
2C–/
– L1
TFAP
2C–/
– L2
1 0.08 0.02
1 0.17 0.07
Cont
rol
TFAP
2C–/
– L1
TFAP
2C–/
– L2
TFAP2C
H3
5d 5iLAFControl
line TFAP2C–/–L1
c
d
Rel
ativ
e A
TA
C e
nric
hmen
t1
2
3
4
5
6
7
−2,000 −1,000 1,000 2,000
Naive ATACd5 5iLAF (WT)
d5 5iLAF TFAP2C –/–
Primed ATACe
Rel
ativ
e C
hIP
enr
ichm
ent
Naive TFAP2C ChIP(dashed line indicates ChIP input)
d5 5iLAF TFAP2C ChIP
1
2
3
4
5
−2,000 −1,000 1,000 2,000Naitive-specificATAC peaks
DAPI TFAP2C OCT4 Merge
Control
TFAP2C –/–
line 1
DAPI PAX6 Mergei j
k
All n
aive
(82,
585
peak
s)
Naive
spec
ific
(5,0
32 p
eaks
)
All p
rimed
(79,
048
peak
s)
Prim
ed sp
ecific
(2,5
62 p
eaks
)
1.0
1.5
2.0
2.5
3.0
Fol
d-en
richm
ent
for
AP
2 m
otifs
AP2 motif enrichmentover ATAC sets (human)
All n
aive
(42,
581
peak
s)
Naive
spec
ific
(2,3
36 p
eaks
)
All p
rimed
(40,
986
peak
s)
Prim
ed sp
ecific
(810
pea
ks)
AP2 motif enrichmentover ATAC sets (mouse)
3.5
1.0
1.5
2.0
2.5
3.0
3.5
* * * *Fold
-enr
ichm
ent
for
AP
2 m
otifs
l
WT
spec
ific
(373
pea
ks)
Tfap
2a–/
– 2c–/
–
spec
ific (1
51
peak
s)
Naive WT versusTfap2a–/– Tfap2c–/–
1.0
1.5
2.0
2.5
3.0
*0.0
0.5
1.0
1.5Tfap2c–/–
Tfap2a–/– Tfap2c–/–
Rel
ativ
e ex
pres
sion
KO
/WT
Tfap
2cOc
t4So
x2
Nano
g
Fol
d-er
nric
hmen
tfo
r A
P2
Mot
ifs
m n
50 kDa 50 kDa
50 kDa
40 kDa
15 kDa
50 kDa
50 kDa
40 kDa 40 kDa
TFAP2C –/–L2
Naitive-specificATAC peaks
Fig. 4 | TFAP2C−/− cells differentiate in naive media. a,
TFAP2C−/− hESCs self-renew in primed conditions but differentiate
and fail to self-renew on treatment in naive (5iLAF) media. Scale
bars, 100 μ m. Data represent one of four independent experiments
with similar results. b, Western blot of TFAP2C upon culture in
primed or 5iLAF conditions. TFAP2C is strongly induced within
3 days of treatment with 5iLAF. Data represent one of two
independent experiments with similar results. c, Western blot for
TFAP2C after 5 days of 5iLAF culture. TFAP2C is absent from
TFAP2C−/−-deficient lines. d, ATAC-seq openness of naive, d5 5iLAF
wild type (WT) and TFAP2C−/−, and primed cells over naive-specific
ATAC peaks. Note that, after 5 days of reversion, substantial
opening of the naive-specific ATAC peaks has already occurred, but
not in the TFAP2C−/− cells. e, TFAP2C ChIP enrichment shown over
naive and d5 5iLAF samples. In e, the ChIP input for each set is
shown as a dashed line. f, Western blot for OCT4 and NANOG in
control and TFAP2C−/− cells after 5 days of culture in 5iLAF.
Quantification is normalized to histone below. g, Western blot for
SOX1 and PAX6 in control and TFAP2C−/− cells. h, Relative RPKM
(reads per kilobase per million mapped reads) of pluripotency and
neural markers in RNA-seq. Data are from n = 3 WT and n = 4
TFAP2C−/− independent biological replicates (mean ± s.e). i,j,
Immunofluorescent staining for TFAP2C, OCT4 (i) and PAX6 (j) in
control and TFAP2C−/− cells. Scale bars, 20 μ m. k,l, Fold
enrichment for AP2 motifs in the specified peak sets in humans (k)
and mouse (l). *No enrichment. Although AP2 motifs are enriched in
naive-specific peaks in both species, the enrichment is much
stronger in the human naive-specific set. m, Expression of key
pluripotency markers in WT, Tfap2c−/− and Tfap2a−/−Tfap2c−/− cells
in 2i + LIF conditions. n = 4 biological replicates for Tfap2c−/−
and controls and n = 6 biological replicates for
Tfap2a−/−Tfap2c−/− and controls (mean ± s.e.) n, ATAC-seq peaks
specific to WT and Tfap2a−/−Tfap2c−/− were calculated, and
enrichment for AP2 motifs was determined. *No enrichment. Uncropped
western blots are provided in Supplementary Fig. 9. Source data for
h and m are provided in Supplementary Table 8.
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We found AP2 and KLF motifs strongly enriched in blastocyst and
naive hESC chromatin, consistent with the reported activation of
AP2 and KLF-family transcription factors in morula and contin-ued
expression in human epiblast and trophoblast (Fig. 2h,i). The GATA
transcription factor motif was strongly enriched in
blasto-cyst-specific chromatin while the OCT4− SOX2 motif was
strongly enriched in naive and primed hESCs, consistent with
preferential expression of GATA2 and GATA3 in the trophoblast and
OCT4 in the inner cell mass (ICM) and epiblast. Our data thus
strongly
support the idea that naive hESCs have an open chromatin state
similar to pre-implantation epiblast.
Using an alternative approach we further confirmed the in vivo
relevance of the naive-specific ATAC peaks by analysing DNA
meth-ylation, given that regulatory elements are typically
hypomethylated relative to the surrounding sequence30,31.
Consistent with this trend, we observe strong hypomethylation of
naive-specific ATAC-seq peaks in naive hESCs cultured in 5iLAF or
in t2iLGö10, a different cul-ture method for generating naive
hESCs6,12 (Supplementary Fig. 2b).
H3
TFAP2C
Cont
rol c
ells
TFAP
2C–/
– line
1
TFAP2C –/ – line 1Dox-induc TFAP2C
TFAP2C –/– line 1Dox-induc TFAP2C
+0 Dox
TFAP2C –/– line 1Dox-induc TFAP2C+0.125 μg ml–1 Dox
TFAP2C –/ – line 1Dox-induc TFAP2C TFAP2C –/ – line 1
Dox-induc TFAP2C
0 0.25 0.50 0.75 1.00 (Dox μg ml–1)
a
d
Controlcells Control
cells
0 (Dox μg ml–1)
OCT4
H3
0.5
b
SOX1
H3
0 0.25 0.50 (Dox μg ml–1)
c
TFAP
2C
POU5
F1
NANO
GSO
X2KL
F4KL
F5
TFCP
2L1
DNM
T3L
OTX2
ZIC2
ZIC3
1/32
1
32
1,0240.125 Dox
0.25 Dox
0.5 Dox
Naive
Exp
ress
ion
rela
tive
topr
imed
con
trol
Corepluripotency
Naivepluripotency
Primedpluripotency
e
f
−2,0
00
−1,0
00
Naive ATACpeak summit
1,00
02,
000
Primed
Control d27 5iLAF
TFAP2C –/– line 1 Dox-induc TFAP2C +0.25 μg ml–1 Dox d27
5iLAFTFAP2C –/– line 1 Dox-induc TFAP2C +0.25 μg ml–1 (Dox
withdrawn d15) d27
−2,0
00
−1,0
00
Naive ATACpeak summit(AP2+ KLF–)
1,00
02,
000
−2,0
00
−1,0
00
Naive ATACpeak summit(AP2– KLF+)
1,00
02,
000
−2,0
00
−1,0
00
Primed ATACpeak summit
1,00
02,
000
Rel
ativ
e A
TA
C e
nric
hmen
t
2
4
6
8
10
12
14
2
4
6
8
10
12
14
2
4
6
8
10
12
14
2
4
6
1
3
5
d19
50 kDa
50 kDa
15 kDa
40 kDa 40 kDa
TFAP2C –/– line 1Dox-induc TFAP2C+0.25 μg ml–1 Dox
TFAP2C –/– line 1Dox-induc TFAP2C+0.50 μg ml–1 Dox
Fig. 5 | Ectopic expression of TFAP2C partially rescues the
TFAP2C−/− phenotype. a, Quantitative western blot showing tunable
TFAP2C induction in TFAP2C−/− background in primed conditions. b,c,
Western blots show rescue of OCT4 expression and SOX1 repression
upon doxycycline-inducible TFAP2C expression. Lysates were
collected after 5 days of treatment with 5iLAF and the indicated
concentration of doxycycline. d, Appearance of round naive-like
colonies in line with ectopic TFAP2C expression. Scale bar, 100 μ
m. Results represent one of four independent experiments with
similar results. e, Partial rescue of upregulation of naive
pluripotency factors, downregulation of primed-factors with ectopic
TFAP2C expression; one replicate for dox induction samples and
primed control, four for naive samples and primed control. f,
TFAP2C was ectopically expressed for the first 15 days of
reversion, then removed in some cells to induce acute loss of
TFAP2C. ATAC-seq plotted from these cells is plotted over
naive-specific peaks (5,032 peaks), a subset that contained an AP2
motif but no KLF motif (1054 peaks), a subset that contained a KLF
motif but no AP2 motif (1,551 peaks), and primed-specific peaks
(2,562 peaks). Reduced ATAC-seq density over naive specific peaks
and increased density over primed-specific peaks, in the sample in
which doxycycline had been withdrawn. Closing of naive specific
peaks is especially pronounced over the subset of peaks that
contain AP2 sites but no KLF sites (AP2+ KLF−). Peak subsets are
listed in Supplementary Table 2. Uncropped western blot images are
provided in Supplementary Fig. 9. Source data for e are provided in
Supplementary Table 8.
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Blastocyst
Control line5d 5iLAF
5iLAF control5% O
2 SSEA4–
5iLAF control5% O
2 SSEA4+
TFAP2C–/–
low O25% SSEA4–
TFAP2C–/–
low O25% SSEA4+
Primed control for low O2
NaiveR1 Naive R2
Naive R3Naive R4
Primed R1Primed R2
Primed R3
Primed control for DoxTFAP2C
–/– L1
d5 5iLAF
TFAP2C–/–
L2d5 5iLAF
Control cells (UCLA1)5d 5iLAF
5iLAF controlSSEA4–
5iLAF controlSSEA4+
TFAP2C–/–
0.25 Dox5iLAF SSEA4–
TFAP2C–/–
0.25 Dox5iLAF SSEA4+
Withdrawn Dox5iLAF SSEA4+
Withdrawn Dox5iLAF SSEA4-
Primed R4
PCA axis 1 (55.3%)
PC
A a
xis
2 (1
6.6%
)
−2,0
00
−1,0
00
Naive
ATA
C
peak
sum
mit
1,00
02,
000
−2,0
00
−1,0
00
Prim
ed A
TAC
peak
sum
mit 1,
000
2,00
0
Rel
ativ
e A
TA
C e
nric
hmen
t
2
4
6
8
1
2
3
4
5
6
Primed
Control line 5% O2 SSEA4–
TFAP2C –/– line 1 5% O2 SSEA4–
TFAP2C –/– line 1 5% O2 SSEA4+
Control line5iLAF 5% O2
TFAP2C –/– line 15iLAF 5% O2
d5
d16
Passage 1 (d10) Passage 1 (d10)
d34
105
104
103
102
105
105
104
104
103
103
102
102105104103102
105104103102105104103102
TRA-1-85-PE
SS
EA
4-A
PC
TRA-1-85-PE
SSEA4-APC SSEA4-APC
Cou
nt
10
20
30
40
10
20
30
Passages 2-4 Passages 2-4
−2,0
00
−1,0
00
Naive
ATA
C
peak
sum
mit
(AP2
+ KL
F–) 1,
000
2,00
0
−2,0
00
−1,0
00
Naive
ATA
C
peak
sum
mit
(AP2
– KL
F+)1,
000
2,00
0
2
4
6
8
2
4
6
8
a c
d
e
b
OCT4
SOX1 H3
PAX6
Control lined5 5iLAF
TFAP2C–/–
line 1d5 5iLAF
Control lined5 5iLAF
TFAP2C–/–
line 1d5 5iLAF
0.0001
0.001
0.01
0.1
1
10
100
1,000
1,000100101
Exp
ress
ion
cont
rol/T
FA
P2C
–/–
/ (5i
LAF
, 5%
O2)
0.1
Expression naive/primed
0.010.0010.0001
40 kDa
40 kDa
50 kDa
15 kDa
85.4 79.7
7.62 86.872.3 17.8
Fig. 6 | TFAP2C−/− cells survive in 5iLAF in 5% o2 conditions
but do not transition to naive state. a, Western blots for the
pluripotency marker OCT4 and the neural markers SOX1 and PAX6 in WT
and TFAP2C−/− cells after 5 days in 5iLAF at 5% O2. b,c,
Bright-field images of control and TFAP2C−/− cells in 5iLAF
culture. Initially the TFAP2C−/− cells show morphology similar to
what is observed under ambient oxygen concentration conditions
(compare to Fig. 4a). However, some colonies are observable after
passaging. These colonies show a shift toward the SSEA4+ (primed)
state. Scale bar, 100 μ m. c, ATAC-seq data from control and
TFAP2C−/− cells in 5% O2 plotted over ATAC-seq peak sets. d,
Principle component analysis comparing ATAC-seq data sets generated
in this work. Blue dots: after 5 days in 5iLAF, WT control cells
show an ATAC-seq landscape part way between primed and naive,
whereas TFAP2C−/− cells show no change toward naive. Green dots:
although TFAP2C−/− cells survive in low oxygen conditions, they
have an ATAC-seq landscape much more similar to primed than naive
cells. Red dots: ectopic doxycycline-dependent expression of TFAP2C
in TFAP2C−/− partially rescues the naive-landscape, and withdrawal
of doxycycline induces a shift towards primed identity. Shown for
comparison are control cells reverted at the same time. e, Genes
differentially regulated in naive versus primed hESCs are plotted.
Note that genes more highly expressed in naive cells are expressed
lower in TFAP2C−/−. The RPKM values correspond to SSEA4− cells in
control (average of two biological replicates) and SSEA4+ in
TFAP2C−/− (average of three biological replicates). Uncropped
western blot images are provided in Supplementary Fig. 9.
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Articles NATure Cell BiOlOgy
TFAP2C dependent regulatory elements(1,560 sites)
Positive regulation byTFAP2C
Negative regulation byTFAP2C
Distance of element to gene
All g
enes
500–
1,00
0 k
200–
500
k
100–
200
k
50–1
00 k
25–5
0 k
10–2
5 k
5–10
k2–
5 k1–
2 k
4 fold downregulatedin TFAP2C–/–
(509 genes)
>4 fold upregulatedin TFAP2C–/–
(1,048 genes)
Identifying genes regulated by TFAP2C
Naive specific ATAC-peaks (5,032 peaks)
Identifying TFAP2C-dependent regulatoryelements
Overlap with TFAP2C ChIP peaks (1,982 sites)
TFAP2C dependent regulatory elements(>50% signal reduction in
TFAP2C–/–
adjusted for read depth (1,560 sites))
−2,000 −1,000 ATAC peaksummit
1,000 2,000
2
4
6
8
Rel
ativ
e A
TA
C e
nric
hmen
t
−2,000 −1,000 ATAC peaksummit
1,000 2,000
2
4
6
8
−2,000 −1,000 ATAC peaksummit
1,000 2,000
2
4
6
8
Prime (control run at same timeas low O2)
Control line low O2 SSEA4–
TFAP2C–/– line 1 low O2 SSEA4–
TFAP2C–/– line 1 low O2 SSEA4+
g
a c e
b
f
h i
d
Chr2:122,000 kb 122,030 kb
TFCP2L1
122,060 kb
Naive ATAC
Primed ATAC
Primed TFAP2C
Blastocyst ATAC
Naive TFAP2C
Primed H3K27Ac
Low O2control ATAC
Low O2TFAP2–/– ATAC
Naive H3K27Ac
200k563
200k104
Rel
ativ
e A
TA
C e
nric
hmen
tR
elat
ive
AT
AC
enr
ichm
ent
Fig. 7 | identifying direct regulatory targets of TFAP2C. a,
Percentage of the time a gene whose TSS is a given distance from a
TFAP2C ChIP-seq peak is upregulated or downregulated in naive
hESCs. Notice the much weaker correspondence compared with Fig. 1a,
and the lack of any effect at the promoter. b, Distance of TFAP2C
ChIP-seq peak to nearest TSS. c, To identify pluripotency-state
genes positively or negatively regulated by TFAP2C, we identified
the subset of naive-specific genes downregulated less than fourfold
in TFAP2C−/− (positively related by TFAP2C) and primed-specific
genes upregulated more than fourfold in TFAP2C−/− (negatively
regulated by TFAP2C). Because they were the predominant pluripotent
populations, we compared expression of SSEA4− control cells (n = 2
biological replicates) to SSEA4+ TFAP2C−/− cells (n = 3 biological
replicates). d, To identify TFAP2C-dependent enhancers, we
identified the overlap of the naive-specific and TFAP2C ChIP-seq
peaks, then took the subset of peaks that showed > 50% density
reduction in TFAP2C−/− SSEA4+ as compared with control SSEA4−
cells, normalized for total read depth. These were classified as
TFAP2C-dependent regulatory elements. e, ATAC-seq read density over
all naive-specific ATAC peaks, naive-specific ATAC peaks
overlapping with TFAP2C ChIP-seq peaks, and the TFAP2C-dependent
regulatory element set identified in d. Note dramatic loss of
signal in TFAP2C−/− over the TFAP2C-dependent set. f, Frequency
with which a gene a given distance from a TFAP2C-dependent ATAC-seq
peak is positively or negatively regulated by TFAP2C. g, Distance
of TFAP2C-dependent ATAC-seq peak to the nearest gene TSS. Note
that the vast majority of such elements are enhancers. h, Schematic
demonstrating the typical regulatory role of TFAP2C in naive hESCs.
Where TFAP2C facilitates the opening of a new enhancer, it has a
positive regulatory role. Where it hones to chromatin that is
already open, it has no tangible effect on transcription. i,
ATAC-seq and ChIP-seq data are shown in the vicinity of
naive-pluripotency factor TFCP2L1.
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Similarly, we observe a more pronounced drop in DNA methyla-tion
between the oocyte and blastocyst stages of human embryonic
development at our defined set of naive-specific ATAC peaks than
over the surrounding sequence or primed-specific ATAC peaks
(Supplementary Fig. 2c,d). Thus, multiple lines of evidence support
the proposition that the majority of putative regulatory elements
identified in naive hESCs correspond to a hypomethylated
regula-tory element in human pre-implantation embryos.
TFAP2C supports reversion to the human naive state. Of the five
AP2-family transcription factors present in humans, only TFAP2C is
highly expressed in the naive state (Fig. 3a). TFAP2C is
upregu-lated in naive cells at both the RNA and protein level (Fig.
3a,b) and is expressed in human morula and pre-implantation
epiblast3,8,32. ChIP for TFAP2C showed strong enrichment over
naive-specific
ATAC-seq peaks (Fig. 3c,d), especially those containing AP2
motifs (Fig. 3d). Furthermore, TFAP2C showed stronger enrichment at
naive-specific ATAC-seq peaks than at regions open in both the
primed and naive state (Fig. 3e), even though both ATAC peak sets
show similar ATAC-seq enrichment in the naive cells (Fig. 3f).
Combined with our observation that AP2 motifs are specifically
enriched in naive-specific peaks, these data indicate that TFAP2C
may facilitate the opening of naive-specific regulatory
elements.
We used CRISPR to target TFAP2C in the primed state, and lines
containing null mutations of both alleles were confirmed
karyotypi-cally normal (Supplementary Fig. 3a,b). In the primed
state these lines showed normal expression of pluripotency genes
and markers (Supplementary Fig. 3c–f). The TFAP2C−/− cells were
able to exit pluripotency normally with spontaneous embryoid body
(EB) dif-ferentiation, and showed a skew towards neural lineage,
consistent
a Chr6:
Oct4 WT (5iLAF)
Oct4 Δintron element 1 (5iLAF)
Oct4 WT (primed)
Oct4 Δintron element 1 (primed)
21
0.25
0.0625
0.0156
0.0039
d5 d14 d19 SortedSSEA4–
Days 5iLAF reversion
Oct
4 ex
pres
sion
(Δ e
nhan
cer/
WT
)
Naive ATAC
Primed ATAC
Primed TFAP2C
Blastocyst ATAC
Naive TFAP2C
Primed H3K27Ac
Naive H3K27Ac
Primed mediator
Naive mediator
Low O2control ATAC
Low O2TFAP2–/– ATAC
POU5F1 (OCT4)
31,132 kb 31,142 kb31,137 kb
Intronelement 1
Proximalenhancer
Distalenhancer
Intronelement 2
Chr6:31,136,000 31,138,00031,137,000
AP2AP2
AP2AP2
AP2AP2 KLF
KLF
KLFKLFKLFGATA
GATAGATAGATA
TEAD
POU5F1 (OCT4)
Intron element 2 Intron element 1
b
Naive ATAC
Primed ATAC
Blastocyst ATAC
Primed TFAP2C
Naive TFAP2C
Low O2control ATAC
Low O2TFAP2–/– ATAC
Targeted region (chr6: 31137320-31137669)
Intro
n
elem
ent 1
Intro
n
elem
ent 1
ΔAP2
0
2
4
6
Luci
fera
se r
elat
ive
to p
GL3
Intro
n
elem
ent 1
ΔKL
F
Chr17:35,640 kb 35,650 kb35,645 kb
2i+LIFmouse(ATAC)
POU5F1 (OCT4)
c f
d e
Fig. 8 | A TFAP2C+ intronic enhancer of oCT4. a, Chromatin
landscape of OCT4. Two putative enhancers, intron element 1 and
intron element 2, are present in blastocyst. Intron element 1 is
also strongly enriched in naive cells and lost in TFAP2C−/−. b, The
location of consensus motifs for key pre-implantation transcription
factors is shown in the vicinity of intron elements 1 and 2. Note
the clustering of AP2 sites at each element. The control low O2
track is the SSEA4− population, the TFAP2C−/− low O2 is the SSEA4+
population. The region targeted for CRISPR deletion is shown. c,
ATAC-seq reads over the murine Pou5f1 locus in naive (2i + LIF)
conditions. Note the absence of either intronic enhancer. d,
Luciferase activity from a pGL3 construct in which WT or mutant
intron element 1 had been cloned, normalized to signal from a pGL3
construct with no enhancer. Results are shown from two independent
experiments, except for the Δ AP2 sample, for which there are n =
3 replicates from two experiments. All signals were first
normalized for Renilla signal. e, OCT4 expression is lost over time
upon reversion of the intron element 1-deleted mutant, indicating
differentiation. Sorting for SSEA4− cells in 5iLAF culture
typically produces a pure population of naive hESCs, but this
population has lost OCT4 expression in the intron element 1-deleted
mutant. Mean of n = 2 technical replicates is shown. f, A line in
which the intron element 1 is deleted appears normal in primed
conditions but fails to yield naive colonies on reversion. Scale
bar, 200 μ m. Images are representative of three independent
reversions. Source data for d are available in Supplementary Table
8.
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Articles NATure Cell BiOlOgywith a known role for TFAP2C in
regulating the formation of neural versus non-neural ectoderm27
(Supplementary Fig. 3g).
Upon reversion in naive 5iLAF medium, we observed a dra-matic
morphological change in the TFAP2C−/− hESC lines (Fig. 4a and
Supplementary Fig. 4a,b). Consistent with this rapidly emerg-ing
phenotype we discovered that at day 3 of reversion, TFAP2C protein
is strongly induced in the control cells, but is absent in the
TFAP2C−/− lines (Fig. 4b,c). TFAP2C ChIP and ATAC-seq at day 5 of
reversion show opening of naive-specific enhancers and enrich-ment
of TFAP2C at these sites, but no opening of these enhancers in
TFAP2C−/− (Fig. 4d,e). Initially, the TFAP2C−/− cells divided
rapidly in 5iLAF, but after one passage, round naive-like colonies
were iden-tified only in controls (Fig. 4a). Instead, sparse
clusters of small cells were observed in the TFAP2C−/− lines after
the first passage that ceased to divide and disappeared from
culture. By day 5 of reversion, the TFAP2C−/− cells showed dramatic
loss of pluripotency factors and upregulation of neural lineage
factors (Fig. 4f–j), a result con-firmed by gene ontology (GO)
analysis33 (Supplementary Fig. 4c). Similarly, ATAC-seq showed a
loss of AP2 and pluripotency tran-scription factor motifs in open
chromatin in the TFAP2C−/− cells after 5 days of reversion, and
instead, a gain of peaks enriched for motifs related to neural
development such as SOX and ZIC (Supplementary Fig. 4d,e).
To confirm that this finding was human specific, we performed
ATAC-seq on murine ESCs (mESCs) cultured in the naive state (2i +
LIF) as well as primed EpiSCs34 (Supplementary Table 4). We
discovered that AP2 sites were enriched in naive-specific open
chromatin in 2i + LIF mESCs. However, the degree of enrichment was
far lower than for human naive cells (Fig. 4k,l). We generated
Tfap2c−/− and Tfap2a−/−Tfap2c−/− mESCs (Supplementary Fig. 4f,g)
and found normal expression of pluripotency markers in 2i + LIF
(Fig. 4m). Furthermore, comparing ATAC-seq in control and
Tfap2a−/−Tfap2c−/− double knockout mESCs, we found only 373
control-specific ATAC-seq peaks, and this set was only moderately
enriched for AP2 sites (Fig. 4n). Thus, AP2 transcription factors
play a more modest role in murine than human naive states.
Withdrawal of TFAP2C in naive state causes shift towards the
primed state. Next, we generated a TFAP2C−/− mutant line capable of
expressing TFAP2C in a doxycycline-dose-dependent manner (Fig. 5a).
Overexpression of TFAP2C in primed media did not result in a
pronounced shift toward naive-gene expression, and TFAP2C primarily
honed to regions of chromatin that were already open in primed
hESCs (Supplementary Fig. 5a,b), arguing that the combi-natorial
activity of multiple factors is necessary for primed to naive
reversion.
We then reverted the TFAP2C−/− Dox-inducible line, using 5iLAF
medium supplemented with various quantities of doxycy-cline.
Induction of TFAP2C rescued the morphological abnormal-ity observed
in the mutant, preserved OCT4 expression, repressed SOX1 induction,
and allowed the formation of colonies with naive morphology (Fig.
5b–e and Supplementary Fig. 5c).
To determine the effect of acute loss of TFAP2C, we cultured
cells in 5iLAF + doxycycline until naive morphology colonies were
apparent (day 15), then switched to media without doxycycline. No
acute phenotype was observed; instead, a gradual loss of cells from
culture occurred (Supplementary Fig. 5d,e). Cells remaining 12 days
after doxycycline withdrawal showed increased staining for the
primed surface marker SSEA4 and closing of naive-specific ATAC
peaks, especially the subset containing AP2 sites but no KLF sites
(Fig. 5f and Supplementary Fig. 5f). These findings indicate that
TFAP2C is essential for maintenance as well as establishment of the
naive state.
TFAP2C−/− in low O2. Because low oxygen conditions can stabilize
the pluripotent state and promote human embryogenesis35, we
conducted
two independent reversions in 5% oxygen. Similar to the results
obtained with reversions under ambient (~20% O2), morpho-logical
differences, loss of OCT4, and gain of SOX1 and PAX6 were all
apparent upon culture in 5iLAF in 5% O2 (Fig. 6a,b and
Supplementary Fig. 6a). However, approximately two weeks after
onset of culture in 5iLAF under 5% O2 conditions, round colonies
appeared in the TFAP2C−/− cultures and these putative colonies were
capable of self-renewal (Fig. 6b and Supplementary Fig. 6a).
However, almost all TFAP2C−/− cells had high SSEA4 surface
expression (Fig. 6a), consistent with primed identity12. The second
reversion featured a substantial population of cells with SSEA4
negative identity, but these cells showed gain of neural and loss
of pluripotency markers, indicating that they were differentiated
rather than naive (see RPKM in Supplementary Table 5). ATAC-seq of
TFAP2C−/− cells persisting in 5iLAF under 5% O2 showed reduced
openness over naive-specific peaks, and increased open-ness over
primed-specific ATAC-seq peaks compared with controls (Fig. 6c).
Moreover, principle component analysis of the ATAC-Seq data sets
showed a closer similarity to primed cells (Fig. 6d), with the
transcriptome of the persisting TFAP2C−/− cells present in 5% O2
shifted towards expression of primed-specific genes (Fig. 6e and
Supplementary Fig. 6b,c). In further support of the finding that
persistent TFAP2C−/− colonies in 5% O2 are more primed-like, we
compared the RNA-seq to published primate RNA-seq6 and found a
global reduction in genes specific to pre-implantation epiblast and
an increase in genes specific to post-implantation epiblast
(Supplementary Fig. 6d,e). Finally, we reverted the TFAP2C−/− in
t2iLGöY naive media36 in 5% O2, and similar to the results in
5iLAF, the TFAP2C−/− cells lacked nuclear KLF17, a marker of naive
cells (Supplementary Fig. 6f,g). In total, these data support an
essential role for TFAP2C in the reversion of primed hESCs to the
naive state.
TFAP2C promotes expression of pluripotency genes. The simple
presence of a transcription factor at a locus does not prove a role
in regulating nearby genes, and we observe 14,367 distinct TFAP2C
peaks throughout the genome (Fig. 7a,b), making it difficult to
discern which binding events are important for gene regulation.
Compared with the striking correlation observed between the
presence of a naive-specific enhancer and upregulation of a nearby
gene (Fig. 1a), we observed only a modest correlation between the
presence of a TFAP2C ChIP peak near a gene and the upregula-tion of
that gene in the naive state or downregulation in TFAP2C−/− (Fig.
7a). To the extent an effect was discernable, the presence of a
TFAP2C peak at an enhancer adjacent to the gene was predictive of
upregulation in the naive state, but the presence of a TFAP2C peak
at a gene TSS had a very little effect on the expression of that
gene, which was surprising given that the promoter is a key site of
gene regulation.
We therefore sought to identify direct targets of TFAP2C by
com-bining RNA-seq, ATAC-seq and ChIP-seq data. First, we looked at
the set of genes specific to the naive or primed state and focused
on the subset that showed more than fourfold changes in expression
in TFAP2C−/− (Fig. 7c). Second, we defined a set of
TFAP2C-dependent regulatory elements: TFAP2C ChIP-seq peaks that
overlapped with naive-specific ATAC peaks and showed reduced
openness in TFAP2C−/− (Fig. 7d,e). We found an extremely strong
relationship between downregulation of a gene in TFAP2C−/− and the
presence of a TFAP2C-dependent regulatory element nearby (Fig. 7f
and Supplementary Table 6). The vast majority of TFAP2C-dependent
regulatory elements did not overlap with a gene TSS and were thus
likely to be enhancers rather than promoters (Fig. 7g). By
contrast, TFAP2C ChIP-seq peaks in regions of openness conserved
between naive and primed state had virtually no predictive effect
on gene expression in TFAP2C−/− (Supplementary Fig. 7a,b). In other
words, the primary effect of TFAP2C in naive hESCs is most likely
to open a discrete set of regulatory elements, mainly enhancers
(Fig. 7h).
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Annotations Tool (GREAT)
analysis37 showed that genes within 50 kb of a TFAP2C-dependent
regulatory element were upregulated in Theiller stage 3 and 4
embryos (morula and early blastocyst) and that mutations of these
genes were associated with abnormal embryogenesis (Supplementary
Tables 6 and 7). Adjacent genes included CBFA2T2, TFCP2L1, KLF5,
SOX2, FGF4, NANOG, DPPA3, DPPA5 and TFAP2C itself (Fig. 7i,
Supplementary Fig. 7c–e and Supplementary Table 7), supporting a
role for TFAP2C in directly promoting the naive pluripotent
program.
An intronic enhancer for OCT4 is active in naive hESCs. One of
the characteristic properties that distinguishes naive and primed
states is different enhancer utilization at POU5F1 (OCT4). In
mouse, the proximal enhancer upstream of Pou5f1 is critical for
expression in the post-implantation epiblast, while the distal
enhancer further upstream drives expression in primordial germ
cells and ICM38. In human pre-implantation blastocyst, however,
neither enhancer appears open, whereas two putative enhancers
appear downstream of the POU5F1 TSS (Fig. 8a). Each of these peaks
contains a cluster of AP2 sites and a KLF site, indicating that
they could be opened by the combinatorial activity of these
transcription factors during pre-implantation development (Fig.
8b). Intron element 1 shows evo-lutionary conservation across
placental mammals (Supplementary Fig. 8a) and is open and enriched
for TFAP2C in naive hESCs (Fig. 8a,b), but is not open in naive
mESC (Fig. 8c). We do not observe any reads emanating from this
element spliced into the OCT4 transcript, ruling out the
possibility that it is actually an alter-native promoter
(Supplementary Fig. 8b). Furthermore, we observe enhancer activity
for this region in a luciferase assay, which is largely eliminated
by the loss of either the AP2 sites or KLF site (Fig. 8d).
To examine the role of this enhancer in naive pluripotency, we
ablated this sequence using CRISPR–Cas9 and confirmed normal
karyotype (Fig. 8b and Supplementary Fig. 8c). We found normal
expression of OCT4 (Supplementary Fig. 8d) and self-renewal in the
primed state, but a dramatic loss of OCT4 expression accompa-nied
by differentiation upon reversion to the naive state (Fig. 8e,f).
This indicates a potential direct role for TFAP2C in regulating the
pluripotency master-regulator OCT4 by binding to a previously
unknown enhancer, which in turn is likely to be important for
pre-implantation OCT4 expression.
DiscussionWe present strong evidence that TFAP2C is critical for
the opening of a set of enhancers in naive hESCs. Furthermore, we
show that most of these enhancers are present in human embryo and
therefore biologically relevant, and are likely to directly
regulate genes critical for human naive pluripotency.
TFAP2C has been implicated in both activation and repression of
target loci39–41, which may explain the limited effect of TFAP2C at
promoters where it is already present. However, the enrichment of
AP2 motifs in naive-specific ATAC peaks, the failure of many of
these enhancers to open in the absence of TFAP2C, and the strong
association between TFAP2C-dependent enhancers and expression of
nearby genes is indicative of a critical role for TFAP2C in
regu-lating gene expression by opening enhancers. TFAP2C is known
to interact with members of the CITED family of proteins, which in
turn recruit the histone acetyltransferase p30042–44, suggesting a
model in which TFAP2C facilitates enhancer opening by pro-moting
histone acetylation. Because TFAP2C is expressed in the morula
before blastocyst formation, it could have a role in reset-ting the
chromatin landscape prior to the establishment of naive
pluripotency, analogous to what happens in the artificial system of
in vitro reversion.
The observation that TFAP2C is critical in naive hESCs in vitro
would lead us to predict that TFAP2C is critical for gene
regulation
in pre-implantation epiblast in vivo. This is surprising in
light of the results in mouse, where TFAP2C is clearly dispensable
for ICM and epiblast specification. Tfap2c homozygous null mice
develop to the blastocyst stage22–24, as do mutants generated using
Tfap2cfl/fl Zp3-Cre in which the maternal Tfap2c transcript is
absent23. Tfap2c-deficient mESCs have been successfully derived
from embryos22,28 and gener-ate viable mice in tetraploid
complementation22, indicating that the gene is non-essential in
ICM. Redundancy with other AP2 factors is unlikely to explain this
non-essential role, as Tfap2a−/−Tfap2c−/− double mutant embryos
also develop an epiblast, and the other AP2 factors are expressed
at very low levels in morula and blastocyst23. The major role for
Tfap2c in mouse pre-implantation embryo devel-opment is the
specification and differentiation of trophoblast, with Tfap2c null
mutant mice dying from placental defects45. Notably, while Tfap2c
is strongly enriched in the trophoblast relative to ICM in mouse
blastocysts, human ICM and pre-implantation epiblast retain high
levels of TFAP2C3,8,32. Tfap2c has also been reported in porcine
ICM46, indicating that loss of Tfap2c from the ICM may be specific
to mice. TFAP2C direct targets in naive hESCs include both genes
general to the pre-implantation embryo as well as genes spe-cific
to epiblast such as CBFA2T2, FGF4 and MEG3.
TFAP2C-dependent regulation of OCT4 may also be different in
mouse and human, as is the role of OCT4 itself. In mice, OCT4 is
essential for pluripotency and for repression of trophoblast genes
in the ICM47. CRISPR ablation of OCT4 in human embryos by con-trast
results in outright failure to form blastocyst or express genes
associated with trophoblast or epiblast lineage48. Thus, OCT4 plays
an essential role in humans as early as morula. Our data are
consis-tent with a model in which OCT4 expression is initially
regulated by TFAP2C and KLF-family transcription factors via the
intronic enhancers, and only later is regulated from the
naive-specific dis-tal enhancer. However, alternative possibilities
cannot be ruled out, such as the distal enhancer being active in
morula and decommis-sioned in trophoblast, which makes up the bulk
of early blastocyst.
Morphological and molecular evidence supports the phenom-enon of
the ‘developmental hourglass’, the idea that the devel-opmental
program is actually most evolutionarily conserved in
mid-embryogenesis, and both early and late stages of development
feature high levels of variation across different species49,50. The
dis-covery of a human-specific naive pluripotency factor fits into
this paradigm, and therefore model organisms may only reveal some
of the story of how human embryos develop.
Received: 22 August 2017; Accepted: 20 March 2018; Published
online: 25 April 2018
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AcknowledgementsThe authors thank the UCLA Broad Stem Cell
Research Center (BSCRC) Flow Cytometry core and the UCLA BSCRC High
Throughput Sequencing Core for technical assistance. W.A.P. was
supported by the Jane Coffin Childs Memorial Fund for Medical
Research and a UCLA BSCRC Postdoctoral Training Fellowship. D.C. is
supported by a UCLA BSCRC Postdoctoral Training Fellowship. W.L. is
supported by the Philip J. Whitcome Fellowship from the UCLA
Molecular Biology Institute and a scholarship from the Chinese
Scholarship Council. Work was funded by R01 HD079546 (ATC) and a
NHMRC project grant APP1104560 (to J.M.P.) and a Sylvia and Charles
Viertel Senior Medical Research Fellowships (to J.M.P.). All work
with human pre-implantation embryos was funded by UCLA BSCRC and
not the National Institute of Health. S.E.J. is a fellow of the
Howard Hughes Medical Institute.
Author contributionsW.A.P., D.C., J.H. R.K., T.J.H., A.L. and
X.L. conducted experiments. W.A.P. and W.L. conducted
bioinformatics analysis. W.A.P. and A.T.C. wrote the manuscript.
J.M.P., S.E.J. and A.T.C. supervised the research.
Additional informationSupplementary information is available for
this paper at https://doi.org/10.1038/s41556-018-0089-0.
Reprints and permissions information is available at
www.nature.com/reprints.
Correspondence and requests for materials should be addressed to
S.E.J. or A.T.C.
Publisher's note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional
affiliations.
© 2018 Macmillan Publishers Limited, part of Springer Nature.
All rights reserved.
NATuRE CELL BioLogy | VOL 20 | MAY 2018 | 553–564 |
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ArticlesNATure Cell BiOlOgyMethodsHuman cell culture. Culturing
of primed and naive hESCs, and reversion from primed to naive
state, were conducted as previously reported12, the only
modification being the inclusion of 1× Primocin (Invivogen) in all
media. All cells were cultured in 5% CO2 and ambient oxygen unless
otherwise indicated. Where indicated, doxycycline was added.
The reversion of primed hESCs to naive t2iLGöY state was
performed as previously described36, generating the t2iLGöY medium
as described in a previous report51. All cell lines were cultured
in a 37 °C, 5% O2 and 5% CO2 for the t2iLGöY reversion
experiments.
Murine cell culture. During routine passage and CRISPR editing,
murine ESCs were cultured in serum + LIF media: 15% Hyclone FBS
(ThermoFisher), 1× penicillin/streptomycin/glutamine
(ThermoFisher), 1× non-essential amino acids (ThermoFisher), 55 μ M
β -mercaptoethanol (ThermoFisher), 1× Primocin (Invivogen) and
1,000 U ml−1 ESGRO LIF (Millipore) in knockout DMEM (ThermoFisher).
Cells were passaged with 0.25% trypsin every 3 days and cultured on
mouse embryonic fibroblast (MEFs). Before all RNA-seq or ATAC-seq
experiments, cells were cultured for at least five passages in 2i +
LIF media: 1× N2 supplement, 1× B27 supplement, 1×
penicillin/streptomycin/glutamine (ThermoFisher), 1× non-essential
amino acids (ThermoFisher), 55 μ M β -mercaptoethanol
(ThermoFisher), 1× Primocin (Invivogen), 3 μ M CHIR99021
(Stemgent), 0.5 μ M PD0325901 (Stemgent) and 1,000 U ml−1 ESGRO LIF
(Millipore) in a 50/50% mixture of DMEM/F12 without HEPES
(ThermoFisher) and neurobasal media (ThermoFisher). Cells passaged
in 2i + LIF were passaged every 3 days with 0.25% trypsin and
plated at 50,000 cells per well onto wells pretreated with
poly-l-ornithine (Sigma) and laminin.
Murine EpiSCs were a gift from P. Tesar and were cultured in
primed hESC medium12. EpiSCs were passaged with 1× collagenase type
IV (Life Technologies) every 3 days.
Collecting cell populations for sequencing experiments. To sort
primed and naive hESCs in the steady state for RNA-seq and
ATAC-seq, TRA-1-85+ SSEA4+ and TRA-1-85+ SSEA4− cells were sorted
as previously described12. For the first replicate of RNA-seq from
day 5 reversion cells, MEFs were removed by twice plating the cells
for 5 min on a gelatinized plate to allow MEFs to attach, but for
the second replicate of RNA-seq and for ATAC-seq, MEFs were removed
by sorting for all TRA-1-85+ cells. The isolated human cells were
then processed for sequencing as discussed in the following.
To separate mESCs or EpiLCs from MEFs, cells were detached with
1× trypsin, quenched, and then washed with 1× FACS buffer, stained
with 1:150 anti-SSEA1 in 1× FACS buffer, then washed and stained
with DAPI immediately before sorting. SSEA1+ SSClo DAPI− cells were
sorted and then used for RNA-seq or ATAC-seq.
For human or murine ChIP experiments, cells were collected using
Accutase, quenched, and washed with 1× PBS before fixation.
EB differentiation. Primed hESCs 7 days after plating were
washed with 1× PBS and treated with 1× collagenase type IV at 37 °C
for 1 h, then removed from the plate with short strokes by a cell
scraper. A 4 ml volume of MEF medium (10% qualified fetal bovine
serum (ThermoFisher), Penicillin/Streptomycin/Glutamine
(ThermoFisher), 1x Primocin (Invivogen) in KnockOut DMEM) was added
to the well and the colonies were allowed to settle in a 50 ml
conical tube. Medium was then aspirated by pipette, and the cells
were resuspended in 3 ml mTESR medium with ROCKi and plated in a
low-attachment six-well plate. At the 24 h time point, the EBs were
transferred into a 50 ml conical tube and allowed to settle. Medium
was removed and replaced with primed hESC medium12 lacking FGF. The
medium was changed again at 72 h and the EBs were collected at the
144 h timepoint.
Embryo isolation and generation. Day 6 vitrified blastocysts
were thawed using Vit Kit-Thaw (Irvine Scientific) according to the
manufacturer's protocol. Embryos were cultured in drops of
continuous single culture medium (Irvine Scientific) supplemented
with 20% serum substitute supplement (Irvine Scientific) under
mineral oil for 2–3 h at 37 °C, 6% CO2 and 5% O2. Embryos with good
morphology were used for ATAC-seq.
Targeting of loci with guide RNA. Guide RNA were designed using
crispr.mit.edu. The highest scoring appropriately situated gDNA
sequences were used, with bases removed from the 5′ end as
necessary so that the guide RNA sequence started with base G. Human
TFAP2C was targeted with the guide sequence GCTTAAATGCCTCGTTAC. The
human OCT4 intronic enhancer was targeted with guides
GGCACCCCTTGTAGAAAGC and GTAATGAGTGACCAGACCCT. Murine Tfap2c was
targeted with the guide sequence GTTACTTTGTACTTCGACG. Murine Tfap2a
was targeted with GGGACTATCGGCGGCACG.
CRISPR editing of hESCs. UCLA1 hESCs52 were cultured for at
least two passages in mTESR1 medium (StemCell Technologies) on
Matrigel (Corning). Cells in exponential growth phase were
collected with Accutase, and 800,000 hESCs were electroporated with
4 μ g plasmid DNA using the CA-137, Primary Cell 3 program
in an Amaxa 4-D Nucleofector X-subunit (Lonza). After
transfection, cells were transferred to a single well of a 24-well
plate containing primed hESC medium12 supplemented with 1× Y-27632
(Stemgent). Before transferring the electroporated cells, the
24-well plate was coated with gelatin and with MEFs. The hESCs were
cultured in MEFs in all later steps.
For generation of the TFAP2C-deficient hESC lines, cells were
passaged with Accutase and plated on 10 cm plates for colony
picking the day after transfection. This resulted in heterogenous
colonies, probably because CRISPR-mediated cleavage continued after
single cells were plated for colony picking, requiring later
subcloning. Pure TFAP2C−/− lines were only generated later by
subcloning. The OCT4 intronic enhancer line was plated with on 10
cm plates 3 days after transfection, and did not require later
subcloning.
To obtain clonal and physically separate colonies, cells were
collected with Accutase and 10,000 cells were plated on 10 cm
plates to allow physically separated colonies to grow. Cells were
fed with primed hESC medium starting 2 days after plating and the
medium was subsequently changed every day. Nine to 11 days after
plating, colonies were scored with a syringe and the pieces were
transferred to a 24-well plate, where they were allowed to grow for
an additional six to seven days in primed hESC medium. Cells were
then split with Accutase. Two-thirds of the material was used for
DNA extraction and screening (see section ‘Screening for
mutations’), the remaining third was plated in primed hESC medium
with ROCKi in a well of a 12-well plate. After two days, the medium
was changed to primed hESC media without ROCKi and the cells were
passaged using normal primed conditions described above in the
‘Cell culture’ sections.
For the TFAP2C−/− lines, a further round of colony picking,
expansion and genotyping was conducted to generate pure knockout
populations. Both TFAP2C−/− lines 1 and 2 were generated from the
same round of transfection of UCLA1 hESCs. Control line 1 was
generated by transfection of UCLA1 hESCs with pMaxGFP plasmid and
no CRISPR construct, with cloning and subcloning performed in
parallel.
CRISPR editing of mESCs. mESCs were plated the day before
transfection at a density of 150,000 cells per well in a six-well
plate for each transfection sample. On the day of transfection,
cells were collected with trypsin, precipitated, and then
resuspended in 2.5 ml of serum + LIF medium (see Murine cell
culture above).
In a separate tube, 5 μ g of DNA (1.43 μ g pmaxGFP (Lonza) +
3.57 μ g CAS9/gDNA construct, 5 μ g pmaxGFP for controls) was
diluted to 375 μ l with Opti-MEM medium (ThermoFisher). In another
separate tube, 12.5 μ l of Lipofectamine 2000 (ThermoFisher) was
combined with 375 μ l Opti-MEM. The Lipofectamine/Opti-MEM solution
was incubated for 5 min, combined with the DNA solution, and
incubated a further 20 min at room temperature. The
DNA/Lipofectamine/Opti-MEM mix was added to the suspended cells and
the cells were rotated for 4 h at 37 °C. Transfected cells were
then spun down, resuspended in fresh serum + LIF medium, and plated
on MEFs.
After 48 h, cells were collected with trypsin and GFP+ cells
were sorted and cultured in 96-well plates on MEFs. Three days
after sorting, the medium was changed. Six days after sorting,
wells with colonies were split with trypsin and split onto 24-well
plates with MEFs. After another 3 days, cells were split again,
with 12.5% of the cells split onto a 24-well plate with MEFs to
propagate the line, 25% split onto a gelatin-treated plate without
MEFs to grow cells for DNA extraction, and the rest frozen to
create stocks. After another 3 days, the cells on gelatin were
collected for DNA extraction.
To obtain pure clonal population, the targeted mESCs were later
subcloned by sorting for individual SSEA1+ cells and plated.
Screening for mutations. DNA was extracted using the Quick gDNA
Miniprep kit and the region containing the targeted allele was
amplified by PCR. To screen human TFAP2C−/− and murine TFAP2C−/−
and TFAP2A−/−TFAP2C−/− mutant lines, the Surveyor Mutant Detection
Kit (IDT) was used to identify point mutants, although some point
mutations in the murine lines were large enough to be apparent by
agarose electrophoresis even without Surveyor cutting. For
targeting of the OCT4 naive enhancer, mutant alleles were
identified based on the reduced size of the targeted region.
To determine the identity of the mutations and confirm clonality
of the targeted lines, several strategies were undertaken. First,
bulk PCR product was subjected to Sanger sequencing, to determine
if there was any visible trace from WT product. Second, PCR product
was cloned into the TopoTA vector and at least eight clones
sequenced to identify the mutations in both alleles and confirm no
WT allele. Third, for human TFAP2C−/− and murine Tfap2c−/−, lack of
protein was confirmed by western blot. For the OCT4 intron element
targeting, clonal deletion was also confirmed both by the lack of a
WT-sized band in the initial screening PCR and by the failure to
amplify with primers internal to the deleted region.
Generation of doxycycline-inducible line. TFAP2C was cloned into
a construct facilitating expression under a doxycycline-inducible
(tetON) promoter, followed by autocleaving ‘2 A’ linker and red
fluorescent protein (RFP) to allow detection. This
tetON-TFAP2C-2A-RFP construct was made by cloning TFAP2C-2A-RFP to
replace the hNANOG in FUW-tetO-lox-hNANOG (Addgene 60849).
Vesicular stomatitis Indiana virus glycoprotein (VSVG)-coated
lentiviruses including
© 2018 Macmillan Publishers Limited, part of Springer Nature.
All rights reserved.
NATuRE CELL BioLogy | www.nature.com/naturecellbiology
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Articles NATure Cell BiOlOgytetON-TFAP2C-2A-RFP and FUW-lox-
M2rtTA were generated in HEK 293T cells. TFAP2C mutant line 1 hESCs
were treated with Accutase to make a single cell suspension in 100
μ l hESC medium with ROCKi with 100,000 single cells. Cells were
transduced with a1:1 ratio of tetON-TFAP2C-2A-RFP and
FUW-lox-M2rtTA and plated in 10 cm dishes at different
concentrations. Individual colonies were picked and genotyped for
tetON-TFAP2C-2A-RFP and FUW-lox-M2rtTA.
Reporter assay. The OCT4 intronic enhancer (hg19 chr6
31,137,269–31,137,697) was amplified and cloned into pGL3 Promoter
vector (Promega). Versions with the three AP2 sites (hg19
31,137,370–31,137,378, 31,137,529–31,137,537 and
31,137,547–31,137,555) or KLF site (hg19 31,137,477–31,137,485)
deleted were synthesized by Genewiz and cloned into pGL3. 200,000
naive UCLA19n (first replicate) or UCLA20n (second replicate) hESCs
were then transfected with 800 ng of either empty pGL3 promoter
vector or one of the three constructs described above, along with
200 ng of pRL-TK Renilla vector as a transfection efficiency
control. We used Amaxa nucleofection with P3 buffer and the program
CA-137 and plated the cells onto a 12-well plate well of MEFs. The
cells were then detached from the well with Accutase and lysed, and
luminescence was detected using the Dual-Glo Luciferase system
(Promega).
Immunostaining. For Fig. 4i,j, immunofluorescence was conducted
as published in ref. 12, using anti-TFAP2C (SantaCruz 8977, 1:100),
anti-OCT4 (sc8628-X, 1:100) and anti-PAX6 (R&D Systems AF8150,
1:100). For Supplementary Fig. 6f, immunostaining was performed as
previously described51. The following primary antibodies were used:
rabbit anti-KLF17 polyclonal (1:500, Sigma HPA024629) and mouse
anti-TRA-1-60 IgM (1:300, BD). The following secondary antibodies
were used: goat anti-rabbit IgG AF555 secondary (1:400,
ThermoFisher) and goat anti-mouse IgM AF488 secondary (1:400,
ThermoFisher).
Western blotting. Western blots and quantitation with the
Odyssey Infrared Imager (Licor) were conducted as described
previously12. Antibodies used include anti-OCT4 (SantaCruz sc8628),
NANOG (R&D Systems AF1997), TFAP2C (SantaCruz sc8977 and Abcam
ab76007), SOX1 (R&D Systems AF3369), SOX2 (R&D Systems
MAB2018) and PAX6 (R&D Systems AF8150). Western signals were
normalized to the signal from anti-H3 antibody (Abcam ab10799 or
Abcam ab1791). All antibodies were used at concentrations of
1:1,000 except Santa Cruz anti-TFAP2C (1:700) and H3 (1:3,000).
RNA isolation and library generation. RNA was isolated using the
RNeasy Mini Kit (Qiagen). A 5–50 ng total RNA input was used to
generate sequencing libraries using the Ovation Ultralow Library
System V2 (Nugen) and then Ovation Rapid Library System (Nugen)
protocols.
ATAC-seq library preparation. In all experiments using cultured
cells, between 25,000 and 50,000 sorted cells were subjected to
ATAC-seq as previously reported53. To perform ATAC-seq on embryos,
the embryos were incubated in the reported ATAC-seq lysis buffer
for 10 min, during which they were vortexed for 10 s every 3–4 min,
after which the protocol was conducted identically to the previous
report.
ChIP protocol and library generation. Cells were fixed with 1%
paraformaldehyde (Sigma) and incubated with rotation for 10 min at
room temperature. The paraformaldehyde was quenched by adding
glycine to a final concentration of 0.14 M and rotated another 10
min at room temperature. The cells were then centrifuged at 735g
for 5 min and then flash-frozen with liquid nitrogen and stored at
− 80 °C until ChIP was conducted.
To lyse the cells for ChIP, cells were thawed and resuspended
with 1 ml lysis buffer (10 mM TrisHCl pH 8.0, 0.25% Triton X-100,
10 mM EDTA, 0.5 mM EGTA, 1× protease inhibitors (Roche) and 1 mM
PMSF), then rotated for 15 min. Nuclei were pelleted by
centrifugation at 1,500g for 5 min at 4 °C. Nuclei were then
resuspended with 1 ml 10 mM TrisHCl pH 8.0, 200 mM NaCl, 10 mM
EDTA, 0.5 mM EGTA, 1× protease inhibitor, 1 mM PMSF and rotated for
10 min. Nuclei were then pelleted and resuspended in 650 μ l 10 mM
TrisHCl pH 8.0, 10 mM EDTA, 0.5 mM EGTA,1× protease inhibitor, 1 mM
PMSF and sonicated in a 12 mm × 12 mm sonication tube (Covaris) in
a Covaris S2 (intensity = 5; cycles per burst = 200; duty cycle =
5%; 8 × (30 s on/30 s off) for 4 min effective sonication). The
sonicated lysate was then centrifuged for 10 min at 14,200g, and
the supernatant retained. 10% of the supernatant was saved as
‘Input’ and the rest was used for ChIP.
Protein A Dynabeads (30 μ l, ThermoFisher) were washed three
times with ChIP buffer (16.7 mM TrisHCl pH 8.0, 0.01% SDS, 1.1
Triton X-100, 1.2 mM EDTA, 167 mM NaCl); each wash consisted of the
addition of 1 ml of buffer and collection of the beads on a
magnetic rack (Diagenode). The 30 μ l of beads were then
resuspended in 650 μ l of ChIP buffer and combined with the ChIP
sample to pre-clear the sample. Beads and chromatin were rotated
for 2 h at 4 °C, and the beads were collected and the supernatant
retained. Anti-TFAP2C antibody (3 μ l, sc-8977) was added to the
ChIP sample. The samples were then rotated overnight at 4 °C.
Protein A Dynabeads (60 μ l) were added to the ChIP samples and
rotated for 2 h at 4 °C. The beads were then washed 2 × 4 min with
500 μ l wash buffer A (50 mM HEPES pH 7.9, 1% TritonX-100, 0.1%
deoxycholate, 1 mM EDTA, 140 mM NaCl), 500 μ l wash buffer B (50 m
HEPES pH 7.9, 0.1% SDS, 1% Triton X100, 0.1% deoxycholate, 1 mM
EDTA, 500 mM NaCl) and 500 μ l TE buffer (10 mM TrisHCl pH 8.0, 1
mM EDTA). Each wash consisted of resuspension in 500 μ l buffer and
rotation at 4 min, followed by collection of beads and removal of
supernatant. DNA was eluted in 100 μ l elution buffer (50 mM
TrisHCl pH 8.0, 1 mM EDTA, 1% SDS) at 65 °C for 10 min in a
ThermoMixer (Eppendorf) shaking at 1,400 r.p.m. The eluant was
collected and the beads were subjected to a second round of elution
with 150 μ l elution buffer.
The ChIP eluants were pooled, and the input sample was diluted
to 250 μ l with elution buffer. The samples were incubated 65 °C
overnight to promote decrosslinking. The samples were then allowed
to cool to room temperature, 15 μ g of RNAse A (Purelink,
ThermoFisher) was added, and the samples were incubated for 30 min
at 37 °C to degrade RNA. Proteinase K (100 μ g) was then added and
the samples were incubated at 56 °C for 2 h. DNA was purified using
a MinElute PCR purification kit (Qiagen).
DNA was sonicated again to 150 bp average fragment size with a
Covaris S2, concentrated with Agencourt AMPure XP beads (Beckman
Coulter) and libraries were generated using the Ovation Ultralow
Library System V2 (Nugen).
Replicates and data pooling. All replicates are listed in
Supplementary Table 1 and are biological replicates except where
otherwise noted. For determination of ChIP or ATAC-seq peaks or
display of ChIP or ATAC data in figures, all reads from a given
condition (for example, d5 human ATAC-seq control samples) were
merged to increase coverage. RNA-seq reads for a given condition
were merged when comparing RPKM across conditions or analysing
splicing but were considered separately when calculating
differentially expressed genes (see next section).
RNA-seq data analysis. RNA-seq data were mapped to hg19 using
Tophat54 and read counts per gene were determined using HTSeq55 as
previously described12. Differentially expressed genes were
calculated using DESeq56, and RPKM values were calculated with a
custom script. Once differentially expressed genes were determined,
they were analysed for GO terms called using GOrilla, which
calculates P values and q values using a hypergeometric test33.
Correlation between changes in gene expression and proximity of
ATAC and ChIP peaks was also calculated by a custom script.
ATAC-seq data analysis. ATAC-seq data were mapped using Bowtie
as previously described53. Peaks were defined in each condition
using the MACS2 callpeaks tool57 with appropriate genome size. To
find peaks specific to one condition (for example, naive specific),
we uses the predictd module of MACS2 to determine the predicted
extension size of each data set being compared, callpeaks for each
data set with the –B and –- no model options and with the extension
size specified as the average of the two samples, and the bgddiff
module using the generated pileup and lambda files with the options
–g 60 –l 120. An eightfold relative enrichment cutoff was used to
define peaks specific to each state, except when comparing murine
WT and Tfap2a−/−Tfap2c−/−, in which a sixfold cutoff was used due
to the relatively small number of peaks different in the two
conditions.
To identify peaks in common between the primed and naive states,
and overlap between different peak sets, we used the Bedtools
intersect tool58.
ChIP-seq data analysis. ChIP-seq data were mapped using Bowtie2
with default settings, and clonal reads were removed using samtools
rmdup. Reads from all replicates for a given condition were merged
and